PROJECT SUMMARY Acute respiratory distress syndrome (ARDS) is a major source of morbidity and mortality in patients ventilated for acute respiratory failure. Despite extensive study, little is known about the pathogenesis of ARDS in humans. One salient finding is that barotrauma or ventilator-associated lung injury (VALI) from high-pressure ventilation is a consistent signal for ARDS risk regardless of the underlying disease. Beyond lung protective (low pressure) ventilation, ARDS lacks specific treatments in large part due to the lack of knowledge about the cellular and molecular mechanisms of pathology. Here, we will take advantage of an established ex vivo ventilation and lung perfusion (EVLP) system to directly assess the initial cellular and molecular responses to VALI in human lung. Importantly, each individual will serve as their own control, achieved by ventilating the right and left lung under high and low pressure, respectively. Some studies will incorporate hyperoxia, elevated PEEP, or live bacteria with VALI to provide even more clinical relevance. Single cell RNA sequencing on the experimental and control lungs following ventilation will provide deep molecular profiles of alveolar cells subjected to barotrauma. This tissue-dissociative analysis will be complemented with multimodal (protein and RNA) and multiplex staining of intact lung tissue using two complementary technologies, to correlate the molecular with spatial and morphological features of pathology. We will also test the capacity of human lung to recover from acute VALI by culturing lung slice cultures at air-liquid interface following ventilation, and how this is impacted by pan and endothelial-specific Wnt inhibition, lipopolysaccharide, and targeted pathway manipulations. In summary, our studies will open a window into the dynamic cellular and molecular responses of human lung in acute lung injury.